Genetic control of axillary bud growth in tobacco plants

ABSTRACT

This disclosure provides a number of sequences involved in axillary bud growth in tobacco, methods of using such sequences, tobacco plants carrying modifications to such sequences or transgenes of such sequences, and tobacco products made from tobacco leaf harvested from such plants.

CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF SEQUENCELISTING

This application is a continuation of U.S. patent application Ser. No.14/875,928, filed Oct. 6, 2015, which claims the benefit of U.S.Provisional Application No. 62/060,473, filed Oct. 6, 2014, both ofwhich are incorporated by reference in their entireties herein. Asequence listing contained in the file named “P34468US01 SL.TXT” whichis 322,059 bytes (measured in MS-Windows®) and created on Oct. 1, 2019,is filed electronically herewith and incorporated by reference in itsentirety.

TECHNICAL FIELD

This disclosure generally relates to tobacco plants.

BACKGROUND

Tobacco is a plant species that exhibits exceptionally strong apicaldominance. Molecular signals from shoot apical meristem mediate ahormonal environment that effectively inhibits axillary bud growth. Uponremoval of the apical meristem (also known as “topping”), the signal islost, enabling the formation of new shoots (or “suckers”) from axillarybuds. Sucker growth results in loss of yield and leaf quality. Suckershave been controlled by manual removal and through the application ofchemicals. Maleic hydrazide (MH) and flumetralin are routinely used ontopped plants to inhibit axillary bud growth (suckering). However, laborand chemical agents to control suckers are very expensive. Control ofaxillary bud growth in tobacco through conventional breeding, mutationbreeding, and transgenic approaches have been a major objective forseveral decades but, to date, successful inhibition has not beenachieved through genetic approaches. Therefore, development of tobaccotraits with limited or no axillary bud growth would result in areduction of the use of chemical agents and would reduce costs and laborassociated with tobacco production.

SUMMARY

A number of nucleotide and polypeptide sequences involved in theformation of axillary bud growth are described herein. Methods of usingsuch sequences also are described. The methods described herein allowfor tobacco plants to be produced that exhibit reduced axillary budgrowth after topping.

In one aspect, a tobacco hybrid, variety, line, or cultivar is providedthat includes plants having a mutation in one or more of the nucleicacids shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 57, 59, 61,63, 65, 69, 71, 73, 75, or 77. In some embodiments, the plants exhibit,and can be selected for, reduced axillary bud growth relative to a plantlacking the mutation.

In one aspect, seed produced by any of the tobacco hybrids, varieties,lines, or cultivars is provided, the seed includes the mutation in theone or more nucleic acids.

In another aspect, a method of making a tobacco plant is provided. Sucha method generally includes the steps of inducing mutagenesis inNicotiana tabacum cells to produce mutagenized cells; obtaining one ormore plants from the mutagenized cells; and identifying at least one ofthe plants that comprises a mutation in one or more of the nucleic acidshaving a sequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,57, 59, 61, 63, 65, 69, 71, 73, 75, or 77. Such a method can furtherinclude identifying at least one of the plants that exhibits reducedaxillary bud growth relative to a plant lacking the mutation.

In some embodiments, mutagenesis is induced using a chemical mutagen orionizing radiation. Representative chemical mutagens include, withoutlimitation, nitrous acid, sodium azide, acridine orange, ethidiumbromide, and ethyl methane sulfonate (EMS). Representative ionizingradiation includes, without limitation, x-rays, gamma rays, fast neutronirradiation, and UV irradiation. In some embodiments, mutagenesis isinduced using TALEN. In some embodiments, mutagenesis is induced usingzinc-finger technology.

In another aspect, a method for producing a tobacco plant is provided.Such a method generally includes the steps of: crossing at least oneplant of a first tobacco line with at least one plant of a secondtobacco line, and selecting for progeny tobacco plants that have themutation. Typically, the plant of the first tobacco line has a mutationin one or more nucleic acids having a sequence shown in SEQ ID NOs: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 57, 59, 61, 63, 65, 69, 71, 73, 75, or 77.In some embodiments, such a method can further include selecting forprogeny tobacco plants that exhibit reduced axillary bud growth relativeto a plant lacking the mutation.

In still another aspect, a tobacco product is provided that includescured leaf from a tobacco plant having a mutation in one or more nucleicacids having a sequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 57, 59, 61, 63, 65, 69, 71, 73, 75, or 77. In some embodiments, thetobacco plant exhibits reduced axillary bud growth relative to leaf froma plant lacking the mutation. In some embodiments, the tobacco plantexhibits reduced MR residue relative to leaf from a plant lacking themutation.

In yet another aspect, a method of producing a tobacco product isprovided. Such a method typically includes providing cured leaf from atobacco plant having a mutation in one or more nucleic acids having asequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 57, 59, 61,63, 65, 69, 71, 73, 75, or 77; and manufacturing a tobacco product usingthe cured leaves. In some embodiments, the tobacco plant exhibitsreduced axillary bud growth relative to cured leaf from a plant lackingthe mutation.

As used herein, a mutation can be a point mutation, an insertion, adeletion, and a substitution.

In one aspect, a transgenic tobacco plant is provided that includes aplant expression vector having a nucleic acid molecule at least 25nucleotides in length and at least 91% sequence identity to a sequenceshown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, or 81. In some embodiments, the nucleicacid molecule has at least 91% sequence identity to a sequence shown inSEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, or 81. In some embodiments, expression of thenucleic acid molecule results in a plant exhibiting reduced axillary budgrowth relative to a tobacco plant not expressing the nucleic acidmolecule.

In another aspect, seed produced by any of the transgenic tobacco plantsdescribed herein is provided, wherein the seed comprises the expressionvector.

In another aspect, a transgenic tobacco plant is provided that includesa heterologous nucleic acid molecule of at least 25 nucleotides inlength that hybridizes under stringent conditions to a nucleic acidsequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 75, 77, 79, or 81. In some embodiments, theheterologous nucleic acid molecule hybridizes under stringent conditionsto a nucleic acid sequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, or 81. Insome embodiments, expression of the heterologous nucleic acid moleculeresults in a plant exhibiting reduced axillary bud growth relative to atobacco plant not expressing the nucleic acid molecule.

In some aspects, seed produced by any of the transgenic tobacco plantsdescribed herein is provided, where the seed comprises the heterologousnucleic acid molecule.

In still another aspect, a method of making a transgenic plant isprovided. Such a method typically includes expressing a transgeneencoding a double-stranded RNA molecule that inhibits expression from anucleic acid sequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 57, 59, 61, 63, 65, 69, 71, 73, 75, or 77, wherein thedouble-stranded RNA molecule comprises at least 25 consecutivenucleotides having 91% or greater sequence identity to a sequence shownin SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 57, 59, 61, 63, 65, 69,71, 73, 75, or 77. In some embodiments, wherein expression of thetransgene results in the plant exhibiting reduced axillary bud growthrelative to a plant not expressing the transgene.

In another aspect, a tobacco product is provided that includes curedleaf from any of the transgenic tobacco plants described herein.

In still another aspect, a method of producing a tobacco product isprovided, the method including providing cured leaf from any of thetransgenic tobacco plants described herein; and manufacturing a tobaccoproduct using the cured leaf.

In yet another aspect, a method of reducing axillary bud growth in atobacco plant is provided. Such a method generally includes introducinga heterologous nucleic acid molecule operably linked to a promoter intotobacco cells to produce transgenic tobacco cells, and regeneratingtransgenic tobacco plants from the transgenic tobacco cells. Typically,the heterologous nucleic acid molecule includes at least 25 nucleotidesin length and has at least 91% sequence identity to a nucleic acidsequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 75, 77, 79, or 81. Such transgenic tobaccoplants exhibit reduced axillary bud growth. In some embodiments, theheterologous nucleic acid molecule has at least 91% sequence identity toa nucleic acid sequence as shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, or 81. Insome embodiments, expression of the nucleic acid molecule results in aplant exhibiting reduced axillary bud growth relative to a tobacco plantnot expressing the nucleic acid molecule. Such a method further caninclude selecting at least one of the transgenic tobacco plants thatexhibits reduced axillary bud growth relative to a tobacco plant notexpressing the heterologous nucleic acid molecule.

In one embodiment, the nucleic acid is in sense orientation. In someembodiments, the nucleic acid is in antisense orientation. In someembodiments, the nucleic acid molecule is introduced into the tobaccocells using particle bombardment, Agrobacterium-mediated transformation,microinjection, polyethylene glycol-mediated transformation,liposome-mediated DNA uptake, or electroporation. In some embodiments,the tobacco plant is a Burley type, a dark type, a flue-cured type, aMaryland type, or an Oriental type. In some embodiments, the tobaccoplant is a variety selected from the group consisting of BU 64, CC 101,CC 200, CC 13, CC 27, CC 33, CC 35, CC 37, CC 65, CC 67, CC 301, CC 400,CC 500, CC 600, CC 700, CC 800, CC 900, CC 1063, Coker 176, Coker 319,Coker 371 Gold, Coker 48, CU 263, DF911, Galpao tobacco, GL 26H, GL 338,GL 350, GL 395, GL 600, GL 737, GL 939, GL 973, GF 157, GF 318, RJR 901,HB 04P, K 149, K 326, K 346, K 358, K394, K 399, K 730, NC 196, NC 37NF,NC 471, NC 55, NC 92, NC2326, NC 95, NC 925, PVH 1118, PVH 1452, PVH2110, PVH 2254, PVH 2275, VA 116, VA 119, KDH 959, KT 200, KT204LC, KY10, KY 14, KY 160, KY 17, KY 171, KY 907, KY907LC, KTY14×L8 LC, LittleCrittenden, McNair 373, McNair 944, msKY 14×L8, Narrow Leaf Madole, NC100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6,NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal SmithMadole, OXFORD 207, ‘Perique’ tobacco, PVH03, PVH09, PVH19, PVH50,PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH51, RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70,Speight H-6, Speight H20, Speight NF3, TI 1406, TI 1269, TN 86, TN86LC,TN 90, TN90LC, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole,Va. 309, or VA359.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the methods and compositions of matter belong. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the methods and compositionsof matter, suitable methods and materials are described below. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety.

DESCRIPTION OF DRAWINGS

FIG. 1A is a graph showing gene expression verification of SEQ ID NO:2using real time PCR analysis. ABO: axillary bud before topping; AB2:axillary bud 2 hr post-topping; AB6: axillary bud 6 hr post-topping;AB12: axillary bud 12 hr post-topping; AB48: axillary bud 48 hrpost-topping; AB72: axillary bud 72 hr post-topping; RT0: roots beforetopping; RT24: roots 24 hr post-topping; YL0: young leaf before topping;YL24: young leaf 24 hr post-topping; ML: mature leaf; MID midrib; SKO:stalk before topping; SK24: stalk 24 hr post-topping; SAM: shoot apicalmeristem and SCL: senescent leaf.

FIG. 1B is a graph showing gene expression verification of SEQ ID NO:19using real time PCR analysis. ABO: axillary bud before topping; AB2:axillary bud 2 hr post-topping; AB6: axillary bud 6 hr post-topping;AB12: axillary bud 12 hr post-topping; AB48: axillary bud 48 hrpost-topping; AB72: axillary bud 72 hr post-topping; RT0: roots beforetopping; RT24: roots 24 hr post-topping; YL0: young leaf before topping;YL24: young leaf 24 hr post-topping; ML: mature leaf; MID midrib; SKO:stalk before topping; SK24: stalk 24 hr post-topping; SAM: shoot apicalmeristem and SCL: senescent leaf.

FIG. 1C is a graph showing gene expression verification of SEQ ID NO:29using real time PCR analysis. ABO: axillary bud before topping; AB2:axillary bud 2 hr post-topping; AB6: axillary bud 6 hr post-topping;AB12: axillary bud 12 hr post-topping; AB48: axillary bud 48 hrpost-topping; AB72: axillary bud 72 hr post-topping; RT0: roots beforetopping; RT24: roots 24 hr post-topping; YL0: young leaf before topping;YL24: young leaf 24 hr post-topping; ML: mature leaf; MID midrib; SKO:stalk before topping; SK24: stalk 24 hr post-topping; SAM: shoot apicalmeristem and SCL: senescent leaf.

FIG. 1D is a graph showing gene expression verification of SEQ ID NO:35using real time PCR analysis. ABO: axillary bud before topping; AB2:axillary bud 2 hr post-topping; AB6: axillary bud 6 hr post-topping;AB12: axillary bud 12 hr post-topping; AB48: axillary bud 48 hrpost-topping; AB72: axillary bud 72 hr post-topping; RT0: roots beforetopping; RT24: roots 24 hr post-topping; YL0: young leaf before topping;YL24: young leaf 24 hr post-topping; ML: mature leaf; MID midrib; SKO:stalk before topping; SK24: stalk 24 hr post-topping; SAM: shoot apicalmeristem and SCL: senescent leaf.

FIG. 1E is a graph showing gene expression verification of SEQ ID NO:37using real time PCR analysis. ABO: axillary bud before topping; AB2:axillary bud 2 hr post-topping; AB6: axillary bud 6 hr post-topping;AB12: axillary bud 12 hr post-topping; AB48: axillary bud 48 hrpost-topping; AB72: axillary bud 72 hr post-topping; RT0: roots beforetopping; RT24: roots 24 hr post-topping; YL0: young leaf before topping;YL24: young leaf 24 hr post-topping; ML: mature leaf; MID midrib; SKO:stalk before topping; SK24: stalk 24 hr post-topping; SAM: shoot apicalmeristem and SCL: senescent leaf.

FIG. 1F is a graph showing gene expression verification of SEQ ID NO:45using real time PCR analysis. ABO: axillary bud before topping; AB2:axillary bud 2 hr post-topping; AB6: axillary bud 6 hr post-topping;AB12: axillary bud 12 hr post-topping; AB48: axillary bud 48 hrpost-topping; AB72: axillary bud 72 hr post-topping; RT0: roots beforetopping; RT24: roots 24 hr post-topping; YL0: young leaf before topping;YL24: young leaf 24 hr post-topping; ML: mature leaf; MID midrib; SKO:stalk before topping; SK24: stalk 24 hr post-topping; SAM: shoot apicalmeristem and SCL: senescent leaf.

FIG. 2 is a schematic of the map of the Agrobacterium transformationvector, p45-2-7.

FIG. 3A shows various nucleic acid alignments (SEQ ID NOs: 1, 13, 33,35, 37, and 55, top to bottom).

FIG. 3B shows various protein alignments (SEQ ID NOs: 2, 14, 34, 36, 38,and 56, top to bottom).

FIG. 4A are photographs of a wild type tobacco plant (left) and atobacco plant transformed with RNA construct #1 (SEQ ID NO:29; right)before topping.

FIG. 4B are photographs of the wild type plant (top) and the planttransformed with RNA construct #1 (bottom) at the indicated time aftertopping.

FIG. 4C are photographs showing the T1 generation produced from thewild-type plant (left) and the plant transformed with RNA construct #1(right).

FIG. 5A shows GUS staining of expression from an axillarymeristem-specific promoter P1 (the promoter from the sequence shown inSEQ ID NO:31) and promoter P7 (SEQ ID NO:32).

FIG. 5B shows GUS staining of expression from promoter P1 (P1:GUSexpression vector) before topping (0 hour) and after topping (24 hr, 48hr and 144 hr).

FIG. 6A are photographs that show the phenotype of the T0 generation fora transgenic line (RNAi_1 (SEQ ID NO:83 against the BRANCH tobaccohomolog); right) in comparison to a wild type plant (left) at 0 h (top)and 1 week after topping (bottom).

FIG. 6B are photographs that show the phenotype of the T0 generation fora transgenic line (RNAi_7 (SEQ ID NO:86 against the BRANCH tobaccohomolog); right) in comparison to a wild type plant (left) at 0 h (top)and 1 week after topping (bottom).

FIG. 6C are photographs that show the phenotype of T1 transgenic plants(RNAi 1; top right, bottom right) in comparison to wild type plants (topleft and bottom left) two weeks after topping.

FIG. 6D is a graph showing that the fresh weight of axillary shoots ofRNAi 1 plants was twice as much as that of wild type plant, indicatingthat silencing the BRANCH1 homolog in tobacco resulted in enhanced budoutgrowth.

FIG. 7A are photographs that show that overexpression of the ArabidopsisBRANCH1 nucleic acid leads to reduced bud outgrowth (right) relative towild type plants (left) within 1 week after topping (bottom).

FIG. 7B are photographs that show that overexpression of the ArabidopsisBRANCH1 nucleic acid influences plant growth in general (right) relativeto wild type plants (left).

FIG. 8 are photographs that show that overexpression of the nucleic acidsequence shown in SEQ ID NO:11 leads to enhanced bud outgrowth aftertopping (right). The phenotype is exemplarily for one transgenic line incomparison to a wild type plant (left) 0 h (top) and 1 week aftertopping (bottom).

FIG. 9 are photographs (close-up, top; entire plant, bottom) that showthat overexpression of RNAi_CET2 in three different transgenic linesdown regulated sucker growth and resulted in reduced bud outgrowth. Thephenotype of three lines transgenic for RNAi CET2 in comparison to awild type plant (left) 1 week after topping is shown.

FIG. 10 are photographs taken 7 days after topping that show thatexpression of RNAi_26 reduced sucker growth (close-up, top right; entireplant, bottom) relative to a wild type plant (top left).

FIG. 11 are photographs showing the meristem-specific expression of GUSunder control of the promoter having the sequence shown in SEQ IDNO:116. As labeled: no expression was observed in the seedling in theabsence of SAM; in the seedling in the presence of SAM, blue color canbe seen; weak expression on axillary buds was seen before topping;strong expression was observed 3 days after topping; GUS expressionfaded out within 5 days after topping; and GUS expression was absent by7 days after topping.

FIG. 12 are photographs showing the axillary bud-specific expression ofGUS under control of the promoter having the sequence shown in SEQ IDNO:117. As labeled: no expression was observed in the seedling in theabsence of SAM; GUS expression was observed in the axillary buds in thepresence of SAM; in the mature plant, GUS expression was observed at thebase and in the side buds; no GUS expression was observed in the flowerbuds; and strong GUS expression was observed in the axillary bud beforetopping and for up to 15 days after topping.

FIG. 13 are photographs showing meristem-specific GUS expression undercontrol of the P1 promoter. GUS expression is observed, but isdown-regulated after topping (at 0 hr).

DETAILED DESCRIPTION

This application describes approaches to produce tobacco with no orreduced sucker growth. For example, the description includes: axillarybud growth gene profiling to discover genes that are critical foraxillary bud development; up regulation of axillary bud growth and/orsucker suppressor genes; down-regulation of axillary bud and/or suckeractivator genes; and modulation of regulatory components of suckergrowth; or initiation or induction of cell death mechanisms in axillarybuds using axillary bud-specific promoters.

This disclosure is based on the discovery of nucleic acids encodingpolypeptides from N. tabacum, Arabidopsis thaliana and Bacillusamyloliquefaciens that are involved in axillary bud growth and theregulation thereof. Such nucleic acids, SEQ ID NOs: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, or 81,and the polypeptides encoded thereby, SEQ ID NOs: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, or 82,are described and characterized herein. Based on this discovery, thelevel of expression of such nucleic acid sequences and/or the functionof such polypeptides can be modulated in Nicotiana species,specifically, for example, N. tabacum. Modulating polypeptide functionand/or gene expression can permit improved control of axillary budgrowth.

Nucleic Acids and Polypeptides

Nucleic acids are provided herein (see, for example, SEQ ID NOs: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, or 81). As used herein, nucleic acids can include DNA and RNA, andincludes nucleic acids that contain one or more nucleotide analogs orbackbone modifications. A nucleic acid can be single stranded or doublestranded, which usually depends upon its intended use. The nucleic acidsprovided herein encode polypeptides (see, for example, SEQ ID NOs: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, or 82).

Also provided are nucleic acids and polypeptides that differ from SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, 75, 77, 79, or 81, and SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, or 82, respectively.Nucleic acids and polypeptides that differ in sequence from SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, or 81, and SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, or 54,can have at least 50% sequence identity (e.g., at least 55%, 60%, 65%,70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, 75, 77, 79, or 81, and SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, or 82, respectively.

In calculating percent sequence identity, two sequences are aligned andthe number of identical matches of nucleotides or amino acid residuesbetween the two sequences is determined. The number of identical matchesis divided by the length of the aligned region (i.e., the number ofaligned nucleotides or amino acid residues) and multiplied by 100 toarrive at a percent sequence identity value. It will be appreciated thatthe length of the aligned region can be a portion of one or bothsequences up to the full-length size of the shortest sequence. It alsowill be appreciated that a single sequence can align with more than oneother sequence and hence, can have different percent sequence identityvalues over each aligned region.

The alignment of two or more sequences to determine percent sequenceidentity can be performed using the computer program ClustalW anddefault parameters, which allows alignments of nucleic acid orpolypeptide sequences to be carried out across their entire length(global alignment). Chenna et al., 2003, Nucleic Acids Res.,31(13):3497-500. ClustalW calculates the best match between a query andone or more subject sequences, and aligns them so that identities,similarities and differences can be determined. Gaps of one or moreresidues can be inserted into a query sequence, a subject sequence, orboth, to maximize sequence alignments. For fast pairwise alignment ofnucleic acid sequences, the default parameters can be used (i.e., wordsize: 2; window size: 4; scoring method: percentage; number of topdiagonals: 4; and gap penalty: 5); for an alignment of multiple nucleicacid sequences, the following parameters can be used: gap openingpenalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes.For fast pairwise alignment of polypeptide sequences, the followingparameters can be used: word size: 1; window size: 5; scoring method:percentage; number of top diagonals: 5; and gap penalty: 3. For multiplealignment of polypeptide sequences, the following parameters can beused: weight matrix: blosum; gap opening penalty: 10.0; gap extensionpenalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro,Ser, Asn, Asp, Gln, Glu, Arg, and Lys; and residue-specific gappenalties: on. ClustalW can be run, for example, at the Baylor Collegeof Medicine Search Launcher website or at the European BioinformaticsInstitute website on the World Wide Web.

Changes can be introduced into a nucleic acid molecule (e.g., SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, 75, 77, 79, or 81), thereby leading to changes in the amino acidsequence of the encoded polypeptide (e.g., SEQ ID NOs: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, or82). For example, changes can be introduced into nucleic acid codingsequences using mutagenesis (e.g., site-directed mutagenesis,transcription activator-like effector nuclease (TALEN), PCR-mediatedmutagenesis, clustered regularly interspaced short palindromic repeats(CRISPR) mutagenesis) or by chemically synthesizing a nucleic acidmolecule having such changes. Such nucleic acid changes can lead toconservative and/or non-conservative amino acid substitutions at one ormore amino acid residues. A “conservative amino acid substitution” isone in which one amino acid residue is replaced with a different aminoacid residue having a similar side chain (see, for example, Dayhoff etal. (1978, in Atlas of Protein Sequence and Structure, 5(Suppl.3):345-352), which provides frequency tables for amino acidsubstitutions), and a non-conservative substitution is one in which anamino acid residue is replaced with an amino acid residue that does nothave a similar side chain.

As used herein, an “isolated” nucleic acid molecule is a nucleic acidmolecule that is free of sequences that naturally flank one or both endsof the nucleic acid in the genome of the organism from which theisolated nucleic acid molecule is derived (e.g., a cDNA or genomic DNAfragment produced by PCR or restriction endonuclease digestion). Such anisolated nucleic acid molecule is generally introduced into a vector(e.g., a cloning vector, or an expression vector) for convenience ofmanipulation or to generate a fusion nucleic acid molecule, discussed inmore detail below. In addition, an isolated nucleic acid molecule caninclude an engineered nucleic acid molecule such as a recombinant or asynthetic nucleic acid molecule.

As used herein, a “purified” polypeptide is a polypeptide that has beenseparated or purified from cellular components that naturally accompanyit. Typically, the polypeptide is considered “purified” when it is atleast 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) by dryweight, free from the polypeptides and naturally occurring moleculeswith which it is naturally associated. Since a polypeptide that ischemically synthesized is, by nature, separated from the components thatnaturally accompany it, a synthetic polypeptide is “purified.”

Nucleic acids can be isolated using techniques routine in the art. Forexample, nucleic acids can be isolated using any method including,without limitation, recombinant nucleic acid technology, and/or thepolymerase chain reaction (PCR). General PCR techniques are described,for example in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler,Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleicacid techniques include, for example, restriction enzyme digestion andligation, which can be used to isolate a nucleic acid. Isolated nucleicacids also can be chemically synthesized, either as a single nucleicacid molecule or as a series of oligonucleotides.

Polypeptides can be purified from natural sources (e.g., a biologicalsample) by known methods such as DEAE ion exchange, gel filtration, andhydroxyapatite chromatography. A polypeptide also can be purified, forexample, by expressing a nucleic acid in an expression vector. Inaddition, a purified polypeptide can be obtained by chemical synthesis.The extent of purity of a polypeptide can be measured using anyappropriate method, e.g., column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

A vector containing a nucleic acid (e.g., a nucleic acid that encodes apolypeptide) also is provided. Vectors, including expression vectors,are commercially available or can be produced by recombinant DNAtechniques routine in the art. A vector containing a nucleic acid canhave expression elements operably linked to such a nucleic acid, andfurther can include sequences such as those encoding a selectable marker(e.g., an antibiotic resistance gene). A vector containing a nucleicacid can encode a chimeric or fusion polypeptide (i.e., a polypeptideoperatively linked to a heterologous polypeptide, which can be at eitherthe N-terminus or C-terminus of the polypeptide). Representativeheterologous polypeptides are those that can be used in purification ofthe encoded polypeptide (e.g., 6×His tag, glutathione S-transferase(GST)).

Expression elements include nucleic acid sequences that direct andregulate expression of nucleic acid coding sequences. One example of anexpression element is a promoter sequence. Expression elements also caninclude introns, enhancer sequences, response elements, or inducibleelements that modulate expression of a nucleic acid. Expression elementscan be of bacterial, yeast, insect, mammalian, or viral origin, andvectors can contain a combination of elements from different origins. Asused herein, operably linked means that a promoter or other expressionelement(s) are positioned in a vector relative to a nucleic acid in sucha way as to direct or regulate expression of the nucleic acid (e.g.,in-frame).

Additionally or alternatively, a vector can include sequences to directhomologous recombination of a nucleic acid (e.g., SEQ ID NOs: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, or 81) into a genome. Representative sequences that can directhomologous recombination of a nucleic acid into a genome are known inthe art and include TALEN sequences (e.g., Cermak et al., 2011, Nuc.Acids Res., 39:e82), CRISPR sequences (Jiang et al., 2013, Nuc. AcidsRes., 41:e188), or zinc-finger nucleases (Guo et al., 2010, J. Mol.Biol., 400:96).

Vectors as described herein can be introduced into a host cell. As usedherein, “host cell” refers to the particular cell into which the nucleicacid is introduced and also includes the progeny of such a cell thatcarry the vector. A host cell can be any prokaryotic or eukaryotic cell.For example, nucleic acids can be expressed in bacterial cells such asE. coli, or in insect cells, yeast or mammalian cells (such as Chinesehamster ovary cells (CHO) or COS cells). Other suitable host cells areknown to those skilled in the art and include plant cells. Many methodsfor introducing nucleic acids into host cells, both in vivo and invitro, are well known to those skilled in the art and include, withoutlimitation, electroporation, calcium phosphate precipitation,polyethylene glycol (PEG) transformation, heat shock, lipofection,microinjection, and viral-mediated nucleic acid transfer.

Nucleic acids can be detected using any number of amplificationtechniques (see, e.g., PCR Primer: A Laboratory Manual, 1995,Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; and U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159;and 4,965,188) with an appropriate pair of oligonucleotides (e.g.,primers). A number of modifications to the original PCR have beendeveloped and can be used to detect a nucleic acid.

Nucleic acids also can be detected using hybridization. Hybridizationbetween nucleic acids is discussed in detail in Sambrook et al. (1989,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Sections 7.37-7.57,9.47-9.57, 11.7-11.8, and 11.45-11.57). Sambrook et al. disclosesuitable Southern blot conditions for oligonucleotide probes less thanabout 100 nucleotides (Sections 11.45-11.46). The Tm between a sequencethat is less than 100 nucleotides in length and a second sequence can becalculated using the formula provided in Section 11.46. Sambrook et al.additionally disclose Southern blot conditions for oligonucleotideprobes greater than about 100 nucleotides (see Sections 9.47-9.54). TheTm between a sequence greater than 100 nucleotides in length and asecond sequence can be calculated using the formula provided in Sections9.50-9.51 of Sambrook et al.

The conditions under which membranes containing nucleic acids areprehybridized and hybridized, as well as the conditions under whichmembranes containing nucleic acids are washed to remove excess andnon-specifically bound probe, can play a significant role in thestringency of the hybridization. Such hybridizations and washes can beperformed, where appropriate, under moderate or high stringencyconditions. For example, washing conditions can be made more stringentby decreasing the salt concentration in the wash solutions and/or byincreasing the temperature at which the washes are performed. Simply byway of example, high stringency conditions typically include a wash ofthe membranes in 0.2×SSC at 65° C.

In addition, interpreting the amount of hybridization can be affected,for example, by the specific activity of the labeled oligonucleotideprobe, by the number of probe-binding sites on the template nucleic acidto which the probe has hybridized, and by the amount of exposure of anautoradiograph or other detection medium. It will be readily appreciatedby those of ordinary skill in the art that although any number ofhybridization and washing conditions can be used to examinehybridization of a probe nucleic acid molecule to immobilized targetnucleic acids, it is more important to examine hybridization of a probeto target nucleic acids under identical hybridization, washing, andexposure conditions. Preferably, the target nucleic acids are on thesame membrane.

A nucleic acid molecule is deemed to hybridize to a nucleic acid but notto another nucleic acid if hybridization to a nucleic acid is at least5-fold (e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,50-fold, or 100-fold) greater than hybridization to another nucleicacid. The amount of hybridization can be quantified directly on amembrane or from an autoradiograph using, for example, a Phosphorlmageror a Densitometer (Molecular Dynamics, Sunnyvale, Calif.).

Polypeptides can be detected using antibodies. Techniques for detectingpolypeptides using antibodies include enzyme linked immunosorbent assays(ELISAs), Western blots, immunoprecipitations and immunofluorescence. Anantibody can be polyclonal or monoclonal. An antibody having specificbinding affinity for a polypeptide can be generated using methods wellknown in the art. The antibody can be attached to a solid support suchas a microtiter plate using methods known in the art. In the presence ofa polypeptide, an antibody-polypeptide complex is formed.

Detection (e.g., of an amplification product, a hybridization complex,or a polypeptide) is usually accomplished using detectable labels. Theterm “label” is intended to encompass the use of direct labels as wellas indirect labels. Detectable labels include enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials.

Plants Having Reduced Axillary Bud Growth and Methods of Making

Tobacco hybrids, varieties, lines, or cultivars are provided that have amutation in one or more nucleic acids described herein (e.g., SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 57, 59, 61, 63, 65, 69, 71, 73, 75,or 77). As described herein, stalks of plants having a mutation in oneor more of the nucleic acids (e.g., SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 57, 59, 61, 63, 65, 69, 71, 73, 75, or 77) can exhibit reducedaxillary bud growth (e.g., compared to stalks of a plant that lacks themutation). In some instances, the nucleic acid having the mutation canbe an endogenous nucleic acid; in some instances, the nucleic acidhaving the mutation can be introduced recombinantly.

As used herein, axillary bud growth (or “suckering”) describes theproduction of lateral buds (or “suckers”) that grow between the leaf andthe stalk after a tobacco plant is topped, as commonly understood in theart. Topping refers to the removal of the stalk apex, including theflowers and up to several adjacent leaves, when the plant is nearmaturity, and results in the loss of apical dominance. Provided axillarybud growth is sufficiently controlled, topping increases the yield andthe value-per-acre as well as results in desirable modifications tophysical and chemical properties of the leaf.

Methods of making a tobacco plant having a mutation are known in theart. Mutations can be random mutations or targeted mutations. For randommutagenesis, cells (e.g., Nicotiana tabacum cells) can be mutagenizedusing, for example, a chemical mutagen, ionizing radiation, or fastneutron bombardment (see, e.g., Li et al., 2001, Plant J., 27:235-42).Representative chemical mutagens include, without limitation, nitrousacid, sodium azide, acridine orange, ethidium bromide, and ethyl methanesulfonate (EMS), while representative ionizing radiation includes,without limitation, x-rays, gamma rays, fast neutron irradiation, and UVirradiation. The dosage of the mutagenic chemical or radiation isdetermined experimentally for each type of plant tissue such that amutation frequency is obtained that is below a threshold levelcharacterized by lethality or reproductive sterility. The number of M₁generation seed or the size of M₁ plant populations resulting from themutagenic treatments are estimated based on the expected frequency ofmutations. For targeted mutagenesis, representative technologies includeTALEN (see, for example, Li et al., 2011, Nucleic Acids Res.,39(14):6315-25) or the use of zinc-finger nucleases (see, for example,Wright et al., 2005, The Plant J., 44:693-705). Whether random ortargeted, a mutation can be a point mutation, an insertion, a deletion,a substitution, or combinations thereof.

As discussed herein, one or more nucleotides can be mutated to alter theexpression and/or function of the encoded polypeptide, relative to theexpression and/or function of the corresponding wild type polypeptide.It will be appreciated, for example, that a mutation in one or more ofthe highly conserved regions would likely alter polypeptide function,while a mutation outside of those conserved regions would likely havelittle to no effect on polypeptide function. In addition, a mutation ina single nucleotide can create a stop codon, which would result in atruncated polypeptide and, depending on the extent of truncation, lossof function. Preferably, a mutation in one of the novel nucleic acidsdisclosed herein results in reduced or even complete elimination ofaxillary bud growth after topping in a tobacco plant comprising themutation. Suitable types of mutations in a coding sequence include,without limitation, insertions of nucleotides, deletions of nucleotides,or transitions or transversions in the wild-type coding sequence.Mutations in the coding sequence can result in insertions of one or moreamino acids, deletions of one or more amino acids, and/ornon-conservative amino acid substitutions in the encoded polypeptide. Insome cases, the coding sequence comprises more than one mutation or morethan one type of mutation.

Insertion or deletion of amino acids in a coding sequence, for example,can disrupt the conformation of the encoded polypeptide. Amino acidinsertions or deletions also can disrupt sites important for recognitionof a binding ligand or for activity of the polypeptide. It is known inthe art that the insertion or deletion of a larger number of contiguousamino acids is more likely to render the gene product non-functional,compared to a smaller number of inserted or deleted amino acids. Inaddition, one or more mutations (e.g., a point mutation) can change thelocalization of the polypeptide, introduce a stop codon to produce atruncated polypeptide, or disrupt an active site or domain (e.g., acatalytic site or domain, a binding site or domain) within thepolypeptide. In addition, a target or signal sequence can be mutated,thereby disrupting or altering the placement of the protein in the cell.

Non-conservative amino acid substitutions can replace an amino acid ofone class with an amino acid of a different class. Non-conservativesubstitutions can make a substantial change in the charge orhydrophobicity of the gene product. Non-conservative amino acidsubstitutions can also make a substantial change in the bulk of theresidue side chain, e.g., substituting an alanine residue for anisoleucine residue. Examples of non-conservative substitutions include abasic amino acid for a non-polar amino acid, or a polar amino acid foran acidic amino acid.

Following mutagenesis, M₀ plants are regenerated from the mutagenizedcells and those plants, or a subsequent generation of that population(e.g., M₁, M₂, M₃, etc.), can be screened for a mutation in a sequenceof interest (e.g., SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 57, 59,61, 63, 65, 69, 71, 73, 75, or 77). Screening for plants carrying amutation in a sequence of interest can be performed using methodsroutine in the art (e.g., hybridization, amplification, combinationsthereof) or by evaluating the phenotype (e.g., detecting and/ordetermining axillary bud growth). Generally, the presence of a mutationin one or more of the nucleic acid sequences disclosed herein (e.g., SEQID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 57, 59, 61, 63, 65, 69, 71, 73,75, or 77) results in a reduction of axillary bud growth in the mutantplants compared to a corresponding plant (e.g., having the same varietalbackground) lacking the mutation.

As used herein, reduced axillary bud growth, also referred to as reducedsucker growth, refers to a reduction (e.g., a statistically significantreduction) in the number of axillary buds, a reduction (e.g., astatistically significant reduction) in the size of the axillary buds(e.g., biomass), and/or a reduction (e.g., a statistically significantreduction) of the impact the axillary buds have on agronomic performance(e.g., yield, quality and overall productivity of the plant) compared toa control plant. The effects can be demonstrated as impeding and/oreliminating axillary bud growth after topping, or reducing and/oreliminating the need for application of chemicals (e.g., MH and/orflumetralin) after topping. As used herein, statistically significantrefers to a p-value of less than 0.05, e.g., a p-value of less than0.025 or a p-value of less than 0.01, using an appropriate measure ofstatistical significance, e.g., a one-tailed two sample t-test.

An M₁ tobacco plant may be heterozygous for a mutant allele and exhibita wild type phenotype. In such cases, at least a portion of the firstgeneration of self-pollinated progeny of such a plant exhibits a wildtype phenotype. Alternatively, an M₁ tobacco plant may have a mutantallele and exhibit a mutant phenotype. Such plants may be heterozygousand exhibit a mutant phenotype due to phenomena such as dominantnegative suppression, despite the presence of the wild type allele, orsuch plants may be heterozygous due to different independently inducedmutations in different alleles.

A tobacco plant carrying a mutant allele can be used in a plant breedingprogram to create novel and useful cultivars, lines, varieties andhybrids. Thus, in some embodiments, an M₁, M₂, M₃ or later generationtobacco plant containing at least one mutation is crossed with a secondNicotiana tabacum plant, and progeny of the cross are identified inwhich the mutation(s) is present. It will be appreciated that the secondNicotiana tabacum plant can be one of the species and varietiesdescribed herein. It will also be appreciated that the second Nicotianatabacum plant can contain the same mutation as the plant to which it iscrossed, a different mutation, or be wild type at the locus.Additionally or alternatively, a second tobacco line can exhibit aphenotypic trait such as, for example, disease resistance, high yield,high grade index, curability, curing quality, mechanical harvesting,holding ability, leaf quality, height, plant maturation (e.g., earlymaturing, early to medium maturing, medium maturing, medium to latematuring, or late maturing), stalk size (e.g., small, medium, or large),and/or leaf number per plant (e.g., a small (e.g., 5-10 leaves), medium(e.g., 11-15 leaves), or large (e.g., 16-21) number of leaves).

Breeding is carried out using known procedures. DNA fingerprinting, SNPor similar technologies may be used in a marker-assisted selection (MAS)breeding program to transfer or breed mutant alleles into othertobaccos, as described herein. Progeny of the cross can be screened fora mutation using methods described herein, and plants having a mutationin a nucleic acid sequence disclosed herein (e.g., SEQ ID NOs: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 57, 59, 61, 63, 65, 69, 71, 73, 75, or 77) canbe selected. For example, plants in the F₂ or backcross generations canbe screened using a marker developed from a sequence described herein ora fragment thereof, using one of the techniques listed herein. Progenyplants also can be screened for axillary bud growth, and those plantshaving reduced axillary bud growth, compared to a corresponding plantthat lacks the mutation, can be selected. Plants identified aspossessing the mutant allele and/or the mutant phenotype can bebackcrossed or self-pollinated to create a second population to bescreened. Backcrossing or other breeding procedures can be repeateduntil the desired phenotype of the recurrent parent is recovered.

Successful crosses yield F₁ plants that are fertile and that can bebackcrossed with a parent line if desired. In some embodiments, a plantpopulation in the F₂ generation is screened for the mutation or variantgene expression using standard methods (e.g., PCR with primers basedupon the nucleic acid sequences disclosed herein). Selected plants arethen crossed with one of the parents and the first backcross (BC₁)generation plants are self-pollinated to produce a BC₁F₂ population thatis again screened for variant gene expression. The process ofbackcrossing, self-pollination, and screening is repeated, for example,at least four times until the final screening produces a plant that isfertile and reasonably similar to the recurrent parent. This plant, ifdesired, is self-pollinated and the progeny are subsequently screenedagain to confirm that the plant contains the mutation and exhibitsvariant gene expression. Breeder's seed of the selected plant can beproduced using standard methods including, for example, field testing,confirmation of the null condition, and/or planting to evaluate axillarybud growth.

The result of a plant breeding program using the mutant tobacco plantsdescribed herein are novel and useful cultivars, varieties, lines, andhybrids. As used herein, the term “variety” refers to a population ofplants that share characteristics which separate them from other plantsof the same species. A variety is often, although not always, soldcommercially. While possessing one or more distinctive traits, a varietyis further characterized by a very small overall variation betweenindividuals within that variety. A “pure line” variety may be created byseveral generations of self-pollination and selection, or vegetativepropagation from a single parent using tissue or cell culturetechniques. A “line,” as distinguished from a variety, most oftendenotes a group of plants used non-commercially, for example, in plantresearch. A line typically displays little overall variation betweenindividuals for one or more traits of interest, although there may besome variation between individuals for other traits.

A variety can be essentially derived from another line or variety. Asdefined by the International Convention for the Protection of NewVarieties of Plants (Dec. 2, 1961, as revised at Geneva on Nov. 10,1972, On Oct. 23, 1978, and on Mar. 19, 1991), a variety is “essentiallyderived” from an initial variety if: a) it is predominantly derived fromthe initial variety, or from a variety that is predominantly derivedfrom the initial variety, while retaining the expression of theessential characteristics that result from the genotype or combinationof genotypes of the initial variety; b) it is clearly distinguishablefrom the initial variety; and c) except for the differences which resultfrom the act of derivation, it confirms to the initial variety in theexpression of the essential characteristics that result from thegenotype or combination of genotypes of the initial variety. Essentiallyderived varieties can be obtained, for example, by the selection of anatural or induced mutant, a somaclonal variant, a variant individualplant from the initial variety, backcrossing, or transformation.

Tobacco hybrids can be produced by preventing self-pollination of femaleparent plants (i.e., seed parents) of a first variety, permitting pollenfrom male parent plants of a second variety to fertilize the femaleparent plants, and allowing F₁ hybrid seeds to form on the femaleplants. Self-pollination of female plants can be prevented byemasculating the flowers at an early stage of flower development.Alternatively, pollen formation can be prevented on the female parentplants using a form of male sterility. For example, male sterility canbe produced by cytoplasmic male sterility (CMS), nuclear male sterility,genetic male sterility, molecular male sterility wherein a transgeneinhibits microsporogenesis and/or pollen formation, orself-incompatibility. Female parent plants containing CMS areparticularly useful. In embodiments in which the female parent plantsare CMS, the male parent plants typically contain a fertility restorergene to ensure that the F₁ hybrids are fertile. In other embodiments inwhich the female parents are CMS, male parents can be used that do notcontain a fertility restorer. F₁ hybrids produced from such parents aremale sterile. Male sterile hybrid seed can be interplanted with malefertile seed to provide pollen for seed-set on the resulting malesterile plants.

Varieties, lines and cultivars described herein can be used to formsingle-cross tobacco F₁ hybrids. In such embodiments, the plants of theparent varieties can be grown as substantially homogeneous adjoiningpopulations to facilitate natural cross-pollination from the male parentplants to the female parent plants. The F₂ seed formed on the femaleparent plants is selectively harvested by conventional means. One alsocan grow the two parent plant varieties in bulk and harvest a blend ofF₁ hybrid seed formed on the female parent and seed formed upon the maleparent as the result of self-pollination. Alternatively, three-waycrosses can be carried out wherein a single-cross F₁ hybrid is used as afemale parent and is crossed with a different male parent. As anotheralternative, double-cross hybrids can be created wherein the F₁ progenyof two different single-crosses are themselves crossed.Self-incompatibility can be used to particular advantage to preventself-pollination of female parents when forming a double-cross hybrid.

The tobacco plants used in the methods described herein can be a Burleytype, a dark type, a flue-cured type, a Maryland type, or an Orientaltype. The tobacco plants used in the methods described herein typicallyare from N. tabacum, and can be from any number of N. tabacum varieties.A variety can be BU 64, CC 101, CC 200, CC 13, CC 27, CC 33, CC 35, CC37, CC 65, CC 67, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC900, CC 1063, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263,DF911, Galpao tobacco, GL 26H, GL 338, GL 350, GL 395, GL 600, GL 737,GL 939, GL 973, GF 157, GF 318, RJR 901, HB 04P, K 149, K 326, K 346, K358, K394, K 399, K 730, NC 196, NC 37NF, NC 471, NC 55, NC 92, NC2326,NC 95, NC 925, PVH 1118, PVH 1452, PVH 2110, PVH 2254, PVH 2275, VA 116,VA 119, KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171,KY 907, KY907LC, KTY14×L8 LC, Little Crittenden, McNair 373, McNair 944,msKY 14×L8, Narrow Leaf Madole, NC 100, NC 102, NC 2000, NC 291, NC 297,NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH129, NC 2002, Neal Smith Madole, OXFORD 207, ‘Perique’ tobacco, PVH03,PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81,RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179,Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, SpeightG-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI1269, TN 86, TN86LC, TN 90, TN90LC, TN 97, TN97LC, TN D94, TN D950, TR(Tom Rosson) Madole, VA 309, or VA359.

In addition to mutation, another way in which axillary bud growth intobacco can be reduced is to use inhibitory RNAs (e.g., RNAi).Therefore, transgenic tobacco plants are provided that contain atransgene encoding at least one RNAi molecule, which, when expressed,silences at least one of the endogenous nucleic acids described herein(e.g., SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 57, 59, 61, 63, 65,69, 71, 73, 75, or 77). As described herein, such transgenic plantsexhibit reduced axillary bud growth (e.g., compared to a plant lackingor not expressing the RNAi).

RNAi technology is known in the art and is a very effective form ofpost-transcriptional gene silencing. RNAi molecules typically contain anucleotide sequence (e.g., from about 18 nucleotides in length (e.g.,about 19 or 20 nucleotides in length) up to about 700 nucleotides inlength) that is complementary to the target gene in both the sense andantisense orientations. The sense and antisense strands can be connectedby a short “loop” sequence (e.g., about 5 nucleotides in length up toabout 800 nucleotides in length) and expressed in a single transcript,or the sense and antisense strands can be delivered to and expressed inthe target cells on separate vectors or constructs. A number ofcompanies offer RNAi design and synthesis services (e.g., LifeTechnologies, Applied Biosystems).

The RNAi molecule can be expressed using a plant expression vector. TheRNAi molecule typically is at least 25 nucleotides in length and has atleast 91% sequence identity (e.g., at least 95%, 96%, 97%, 98% or 99%sequence identity) to one of the nucleic acid sequences disclosed herein(e.g., SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 57, 59, 61, 63, 65,69, 71, 73, 75, or 77) or hybridizes under stringent conditions to oneof the nucleic acid sequences disclosed herein (e.g., SEQ ID NOs: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 57, 59, 61, 63, 65, 69, 71, 73, 75, or 77).Hybridization under stringent conditions is described above.

Further, certain of the sequences described herein can be overexpressedin plants to reduce axillary bud growth. Accordingly, transgenic tobaccoplants are provided that are transformed with a nucleic acid moleculedescribed herein (e.g., SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, or 81) or a functionalfragment thereof under control of a promoter that is able to driveexpression in plants. As discussed herein, a nucleic acid molecule usedin a plant expression vector can have a different sequence than asequence described herein, which can be expressed as a percent sequenceidentity (e.g., relative to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, or 81) or based onthe conditions under which the sequence hybridizes to SEQ ID NOs: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, or 81.

As an alternative to using a full-length sequence, a portion of thesequence can be used that encodes a polypeptide fragment having thedesired functionality (referred to herein as a “functional fragment”).When used with respect to nucleic acids, it would be appreciated that itis not the nucleic acid fragment that possesses functionality but theencoded polypeptide fragment. Based on the disclosure herein and thealignments shown in FIG. 3, one of skill in the art can predict theportion(s) of a polypeptide (e.g., one or more domains) that may impartthe desired functionality.

Promoters that drive expression of a coding sequence in plants are knownin the art. Representative promoters include, for example, the CaMV 35Spromoter, the actin promoter, the ubiquitin promoter, the phaseolinpromoter, a rubisco promoter, the zein promoter, an ACEI systempromoter, the In2 promoter, or the H3 histone promoter. In addition,tissue- or developmentally-specific promoter sequences related toaxillary bud growth are described herein and can be used to express oroverexpress a nucleic acid coding sequence. Representative tissue-ordevelopmentally-specific promoter sequences related to axillary budgrowth are shown in SEQ ID NOs: 113, 114, 115, 116, 117, or 118. Asdescribed herein, the coding sequence can be any of the nucleic acidcoding sequences described herein (e.g., SEQ ID NOs: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, or 81);alternatively, a coding sequence can be derived from a gene that resultsin programmed cell death (e.g., nucleic acid molecules that encode aribosome inactivating protein, nucleic acid molecules that encodeproteins involved in the hypersensitive response plants initiate whenconfronted with a pathogen (e.g., a fungus or a bacteria)). Simply byway of example, a tissue- or developmentally-specific promoter sequencerelated to axillary bud growth as described herein can be used toexpress or overexpress a coding sequence whose expression is decreasedafter topping or a coding sequence involved in apoptosis.

Methods of introducing a nucleic acid (e.g., a heterologous nucleicacid) into plant cells are known in the art and include, for example,particle bombardment, Agrobacterium-mediated transformation,microinjection, polyethylene glycol-mediated transformation (e.g., ofprotoplasts, see, for example, Yoo et al. (2007, Nature Protocols,2(7):1565-72)), liposome-mediated DNA uptake, or electroporation.Following transformation, the transgenic plant cells can be regeneratedinto transgenic tobacco plants. As described herein, expression of thetransgene results in a plant that exhibits reduced axillary bud growthrelative to a plant not expressing the transgene. The regeneratedtransgenic plants can be screened for axillary bud growth, and plantshaving reduced axillary bud growth, compared to a correspondingnon-transgenic plant, can be selected for use in, for example, abreeding program as discussed herein.

In addition to overexpression or downregulation of axillary budgrowth-related coding sequences, axillary bud growth can be controlledusing any of the following approaches:

-   -   a. altering the expression of axillary bud growth-related        regulatory genes that are critical for axillary bud development        (as described in Examples 7 and 8);    -   b. altering meristem development-specific genes using axillary        bud-specific promoters;    -   c. altering the hormonal signaling leading to axillary shoot        growth inhibition. This can be accomplished through        overexpression or downregulation of hormonal synthesis or        transport genes driven by tissue specific or timing specific        (e.g., after topping) promoters; and    -   d. initiating cell death mechanisms in axillary buds using        axillary bud specific promoters driving cell suicide or toxicity        genes.

Nucleic acids that confer traits such as herbicide resistance (sometimesreferred to as herbicide tolerance), insect resistance, or stresstolerance, can also be present in the novel tobacco plants describedherein. Genes conferring resistance to a herbicide that inhibits thegrowing point or meristem, such as an imidazolinone or a sulfonylurea,can be suitable. Exemplary genes in this category encode mutant ALS andAHAS enzymes as described, for example, in U.S. Pat. Nos. 5,767,366 and5,928,937. U.S. Pat. Nos. 4,761,373 and 5,013,659 are directed to plantsresistant to various imidazolinone or sulfonamide herbicides. U.S. Pat.No. 4,975,374 relates to plant cells and plants containing a geneencoding a mutant glutamine synthetase (GS), which is resistant toinhibition by herbicides that are known to inhibit GS, e.g.phosphinothricin and methionine sulfoximine. U.S. Pat. No. 5,162,602discloses plants resistant to inhibition by cyclohexanedione andaryloxyphenoxypropanoic acid herbicides.

Genes for resistance to glyphosate also are suitable. See, for example,U.S. Pat. Nos. 4,940,835 and 4,769,061. Such genes can confer resistanceto glyphosate herbicidal compositions, including, without limitation,glyphosate salts such as the trimethylsulphonium salt, theisopropylamine salt, the sodium salt, the potassium salt and theammonium salt. See, e.g., U.S. Pat. Nos. 6,451,735 and 6,451,732. Genesfor resistance to phosphono compounds such as glufosinate ammonium orphosphinothricin, and pyridinoxy or phenoxy propionic acids andcyclohexones also are suitable. See, e.g., U.S. Pat. Nos. 5,879,903;5,276,268; and 5,561,236; and European Application No. 0 242 246.

Other suitable herbicides include those that inhibit photosynthesis,such as a triazine and a benzonitrile (nitrilase). See U.S. Pat. No.4,810,648. Other suitable herbicides include 2,2-dichloropropionic acid,sethoxydim, haloxyfop, imidazolinone herbicides, sulfonylureaherbicides, triazolopyrimidine herbicides, s-triazine herbicides andbromoxynil. Also suitable are herbicides that confer resistance to aprotox enzyme. See, e.g., U.S. Pat. No. 6,084,155 and US 20010016956.

A number of genes are available that confer resistance to insects, forexample, insects in the order Lepidoptera. Exemplary genes include thosethat encode truncated Cry1A(b) and Cry1A(c) toxins. See, e.g., genesdescribed in U.S. Pat. Nos. 5,545,565; 6,166,302; and 5,164,180. Seealso, Vaeck et al., 1997, Nature, 328:33-37 and Fischhoff et al., 1987,Nature Biotechnology, 5:807-813. Particularly useful are genes encodingtoxins that exhibit insecticidal activity against Manduca sexta (tobaccohornworm); Heliothis virescens Fabricius (tobacco budworm) and/or S.litura Fabricius (tobacco cutworm).

Tobacco Products and Methods of Making

Leaf from tobacco plants having reduced axillary bud growth can becured, aged, conditioned, and/or fermented. Methods of curing tobaccoare well known and include, for example, air curing, fire curing, fluecuring and sun curing. Aging also is known and typically is carried outin a wooden drum (e.g., a hogshead) or cardboard cartons in compressedconditions for several years (e.g., 2 to 5 years), at a moisture contentof from about 10% to about 25% (see, for example, U.S. Pat. Nos.4,516,590 and 5,372,149). Conditioning includes, for example, a heating,sweating or pasteurization step as described in US 2004/0118422 or US2005/0178398, while fermenting typically is characterized by highinitial moisture content, heat generation, and a 10 to 20% loss of dryweight. See, e.g., U.S. Pat. Nos. 4,528,993, 4,660,577, 4,848,373 and5,372,149. The tobacco also can be further processed (e.g., cut,expanded, blended, milled or comminuted), if desired, and used in atobacco product.

Tobacco products are known in the art and include products made orderived from tobacco that are intended for human consumption, includingany component, part, or accessory of a tobacco product. Representativetobacco products include, without limitation, smokeless tobaccoproducts, tobacco-derived nicotine products, cigarillos, non-ventilatedrecess filter cigarettes, vented recess filter cigarettes, cigars,snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco,leaf tobacco, shredded tobacco, and cut tobacco. Representativesmokeless tobacco products include, for example, chewing tobacco, snuff,long-cut moist smokeless tobacco, snus, pouches, films, tablets, coateddowels, rods, and the like. Representative cigarettes and other smokingarticles include, for example, smoking articles that include filterelements or rod elements, where the rod element of a smokeable materialcan include cured tobacco within a tobacco blend. In addition to thetobacco described herein, tobacco products also can include otheringredients such as, without limitation, binders, plasticizers,stabilizers, and/or flavorings. See, for example, US 2005/0244521; US2006/0191548; US 2012/0024301; US 2012/0031414; and US 2012/0031416 forexamples of tobacco products.

The invention will be further described in the following examples, whichdo not limit the scope of the methods and compositions of matterdescribed in the claims.

EXAMPLES Example 1—Sampling, RNA Preparation and Sequencing

Tobacco seeds from TN90, a Burley variety, were germinated. After 4weeks, seedlings were transferred onto 4 inch pots. At layby stage (8-10fully expanded leaves), a total of 10 different samples includingaxillary buds before topping (Aa), axillary buds after topping (Ab, Ac,Ad and Ae (2 h, 6 h, 24 h and 7 2h, respectively), roots before topping(Ra), roots after topping (Rb, Rc (24 h and 72 h)), young leaf at thetime of topping (YL), and shoot apical meristem (ST) were collected fornext generation sequencing analysis. Each of the time points wererepresented by three independently collected samples. These threesamples served as biological replicates.

RNA from the samples described above was isolated using RNeasy PlantMini Kit (Qiagen, MA) and quality was tested using Agilent Plant RNANano Kit and a 2100 Bioanalyzer (Agilent Technologies, CA). Thirty cDNAlibraries were constructed, with indexing using a TrueSeq RNA LibraryPrep Kit v.2 (Illumina). cDNA libraries made from the same biologicalreplicates were pooled together, and each pooled replicate was analyzedon an Illumina HiSeq 2000, 100 bp single reads with a minimum of 30million reads per sample. Two samples were tagged per lane for a totalof 15 sequencing lanes. Axillary bud specific gene expression in TN90tobacco was determined by RNA deep sequencing performed by ArrayXpress(Raleigh, N.C.).

Example 2—RNA Sequence Analysis

Gene expression data from five axillary buds, 3 roots, and one each ofyoung leaf and shoot apical meristem samples were analyzed to identifyaxillary bud development-related genes compared to other tissues. Genereads were mapped to our in-house tobaccopedia genome database (Table1). EdgeR in CLC genomic workbench was used to perform differential geneexpression. Gene expression data was filtered for axillary bud specificexpression from other tissues. FDR adjustment was performed on allp-values and a cut-off of an FDR corrected p-value <0.05 was used.Results were then filtered for high axillary bud expression. The list ofdifferentially expressed candidate genes for sucker control are listedin Table 2.

TABLE 1 Mapping of Next Generation Sequencing Reads Using In-HouseTobaccopedia Database Reads Samples Mapped % mapped Aa1 23,920,938 92.03Aa2 49,392,444 91.21 Aa3 28,288,803 86.23 Ab1 24,848,558 92.2 Ab235,727,478 92.23 Ab3 34,000,094 92.25 Ac1 45,951,075 92.04 Ac248,242,863 92.15 Ac3 41,733,418 91.67 Ad1 33,474,960 92.08 Ad231,891,377 92.35 Ad3 40,791,919 92.23 Ae1 28,758,337 92.04 Ae238,369,793 92.26 Ae3 40,552,134 92.45 Ra1 39,732,686 92.02 Ra240,262,611 91.16 Ra3 33,248,092 92.13 Rb1 35,937,062 93.06 Rb240,036,265 92.43 Rb3 46,268,788 92.34 Rc1 35,595,122 92.84 Rc237,925,157 92.25 Rc3 34,832,062 92.18 ST1 48,115,555 92.45 ST241,373,361 92.41 ST3 31,760,672 91.85 YL1 41,811,850 92.63 YL251,356,432 91.82 YL3 40,252,190 91.95

TABLE 2 Differential gene expression of selected candidate genesAxxilary Buds Roots Roots After Before Axillary Buds After ToppingBefore Topping Shoot Contig Topping 2 hr 6 hr 24 hr 72 hr Topping 24 hr72 hr Apical Young Number (AB0) (AB2) (AB6) (AB24) (AB72) (RT0) (RT24)(RT72) Meristem Leaf C5787 1,072 998 1,346 663 652 7 9 11 180 47 C162491,387 927 3,527 44,790 23,270 108 90 128 8,913 72 C3898 763 1,132 1,8525,559 2,644 110 156 80 513 7 C2231 115 532 446 252 496 27 7 11 23 14C49345 2,342 2,357 2,992 3,143 2,190 38 28 27 26 103 C64393 47 29 54 1817 1 0 0 23 1 C26207 128 131 187 69 54 0 1 1 13 0 C83090 124 308 1,619337 136 217 143 160 88 234 C29909 3 162 186 9 9 22 22 29 6 2 C82570 4198 334 136 101 1 0 0 50 0 C12866 1,479 1,486 4,216 16,176 12,228 46 3633 2,144 839 C34805 52 27 81 13 9 2 1 3 5 1 C47069 152 114 135 46 45 2 22 1 0 C73141 60 34 22 17 13 2 4 1 30 1 C41568 176 131 385 48 43 14 12 1519 10 C50303 624 583 1,279 300 215 14 9 18 71 9 C58496 176 121 253 95 707 1 1 69 27 C68375 268 279 410 231 207 1 1 1 22 11 C55919 193 241 366117 123 2 2 2 13 1 C40016 394 353 505 207 204 2 2 1 34 2 C145337 2,1102,953 8,542 1,362 2,095 337 181 337 305 131 C348 1,022 1,253 2,580 715762 79 53 59 164 13 C131180 1,517 2,212 5,081 2,402 1,059 1,109 488 332558 351 C22266 222 265 479 290 187 2 3 1 20 3 C53803 1,796 1,308 3,662777 968 23 21 22 475 11 C21860 104 75 68 107 46 0 1 0 3 0 C11320 486 3091,297 291 395 146 56 42 84 8 C1838 364 175 126 152 97 1 0 0 36 14

Example 3—Confirmation of Selected Candidate Gene Expression

To confirm the expression pattern of selected candidate genes, therelative changes in transcripts from 10-16 different tissue samples (6axillary bud samples (before topping and 2 hr, 6 hr, 12 hr, 24 hr and 72hr after topping), young leaf 24 hr after topping, mature leaf,senescence leaf, midrib, stalk before topping, stalk 24 hr aftertopping, shoot apical meristem, root before topping and 24 hr aftertopping) were measured. In brief, total RNA was isolated using TRIReagent (Sigma-Aldritch, St. Louis, Mo.). To remove DNA impurities,purified RNA was treated with RNase-free DNase (Turbo DNA-free, Ambion,Austin, Tex.). To synthesize the first cDNA strand, approximately 10 μgof total RNA was transcribed utilizing the High Capacity cDNA Kit(Applied Biosystems, Foster City, Calif.). To measure the level ofselected gene transcripts in the samples, RT-PCR was performed usingSYBR Green PCR Master Mix (Applied Biosystems, Foster City, Calif.) andthe gene specific primers listed in Table 3. Real time gene expressionverification of representative candidate genes are listed in FIGS.1A-1G.

TABLE 3Real time PCR Primers used for the confirmation of gene expressionPrimer SEQ SEQ Amplicon Name Forward primer ID NO Reverse primer ID NOsize SCRT1 TTTTCGAGGCTCCTTTAGCA 123 CATGTTGGGGTTCGATAAGG 124 250 SCRT2CCTTTTTTACTCATTCAGAGAAACGA 125 GTGTGACACTGAATTAATCCTTTCC 126 380 SCRT3AGGCTTGCTGAAGCAAAAGA 127 TCGGCGAAATTACAGTCTCA 128 211 SCRT4TTGTGTCATGGTGCAATCAA 129 TCCAACTTAGGCCTCACACC 130 199 SCRT5TTGCAATGCTTCTGTTTTCG 131 ATATTGGCCGCATCTTGGT 132 193 SCRT6TTCTCTTCCCGAGAAACAGTG 133 CGGAGTTGGAGATGAAGATGA 134 217 SCRT7cCTGTGGCAAAGGAATCAAG 135 TGCGTGGTGTGTTCTTCAAT 136 200 SCRT8GGGTGCTTTGAAGTCCCTTT 137 GAATCCTGCTCCAAACAAGC 138 211 SCRT9TGGGCAGCAGAAATAAGAGA 139 GCTGATCTTGTTGTGGCTTG 140 200 SCRT10CACCATAAGCACAGGTGCAA 141 TCCGCCTTGCTTTATGAAAA 142 205 SCRT11TCCTCTTTGCCATTTCTCTCA 143 GGCCAGAAAAAGAATGACCA 144 201 SCRT12GGGTCCCTCTAAATCCCAAG 145 cCGGAAGTCAAGAATCCAGT 146 201 SCRT13TGGACATGAGGCATTTGCTA 147 GCATCGCGAGATCAAGAGTT 148 183 SCRT14AAGCCCGCCTTTCTACCTTA 149 TCTTGATCATCGAACGAATCAC 150 196 SCRT15CCAATTCCCTCTTCCTTCCT 151 ATCCATCCAAGTCAGCCTTC 152 203 SCRT16TGGTTGAGGCCCCAATATAC 153 CCCCGCTATCGACTTGATTA 154 198 SCRT17CGGAAGAGCCTGTGGTATGA 155 TGAAATCAGATTCAGGCATCA 156 203 SCRT18AGATCAGGAAGCGCGTAAGA 157 CAGAGTTTTGCTGGCCTTCT 158 193 SCRT19GTGGCAAAGGAATCAAGGAA 159 ATGGGTTCCAGTTGCCAGTA 160 283 SCRT20CGGTCCTTTAGCAGTTTCCA 161 CATGTTGGGGTTCGATAAGG 162 250 SCRT21ATCTGGAGTATTTCTTCTACCT 163 CTTAAACTCTCTGCCGAATAAA 164 111 SCRT22TCCTTCTTTCTGTCTGTTTCTCTT 165 GTCCTCACTGCTGTCTTTCTC 166 110 SCRT23GCACTTCTGGTGGTGAAAGA 167 GTCATTCTCAGTTATGTTACGGAAAG 168 102 SCRT24AGCTGCTCCATAACCGAAAT 169 CGACCCTGAATTTCCTCTAGTT 170 108 SCRT25GGATGTAAGGCATTGGACATAGA 171 GAGTTCCCTATCAACCGAAACA 172 96 SCRT26GGCGAGTCATTAACCTCCTATTT 173 GTCTTAGCGTCCAAGTGCTAAT 174 117 SCRT27GCTGAAGAACCTTTGCCTTTAC 175 GCCGATTTCTCAACACAAAGAA 176 106

Example 4—Full Length Candidate Genes Cloning, Analysis and SelectedReal Time PCR for Verification

The candidate genes predicted to be involved in axillary bud initiationand growth were identified and annotated (Table 4), and RNAs fromaxillary bud tissues of TN90 plants, from before topping, and 12 hr, 24hr and 48 hr after topping, were collected. cDNA libraries were createdfrom the RNAs using the In-Fusion SMARTER Directional cDNA LibraryConstruction Kit from Clontech (Cat #634933). Full length candidategenes were cloned using the gene specific primers designed frompredicted full-length cDNA sequences. The full length coding sequenceswere confirmed by sequencing and are shown in SEQ ID NOs:1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 57, 59, or 69.The predicted protein sequences are shown in SEQ ID NOs:2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 58, 60, or 70.

TABLE 4 Selected Candidate Genes Contig Nucleo- Protein SEQ ID NO NumberCoding Sequence tide (bp) (aa) (DNA/protein) C5787 Full length confirmed987 328 1/2 C16249 Full length confirmed 318 105 3/4 C3898 Full lengthconfirmed 1797 598 5/6 C7651 Full length confirmed 1392 463 7/8 C49345Full length confirmed 405 134  9/10 C64393 Full length confirmed 630 20911/12 C26207 Full length confirmed 1143 380 13/14 C83090 Full lengthconfirmed 915 304 15/16 C29909 Full length confirmed 1353 450 17/18C82570 Full length confirmed 732 243 19/20 C12866 Pseudo gene — — —C34805 Full length confirmed 471 156 21/22 C47069 Full length confirmed1437 478 23/24 C73141 Full length confirmed 645 214 25/26 C41568 Fulllength confirmed 2205 734 27/28 C50303 Full length confirmed 1302 43329/30 C58496 Full length confirmed 1266 421 31/32 C68375 Full lengthconfirmed 597 198 33/34 C55919 Full length confirmed 1038 345 35/36C40016 Full length confirmed 1014 337 37/38 G47965 Full length confirmed1659 553 57/58 G88345 Full length confirmed 1632 544 59/60 S10610 Fulllength confirmed 396 132 69/70

From RNA sequence analysis and RT-PCR confirmation, candidate putativefull length gene sequences were selected for RNAi and full lengthAgrobacterium transformation analysis. The candidate sequences arelisted in Table 5 and are shown in SEQ ID NOs:39, 41, 43, 45, 47, 49,51, 53, 61, 63, 65, 71, 73, 75, or 77. The predicted protein sequencesare shown in SEQ ID NOs:40, 42, 44, 46, 48, 50, 52, 54, 62, 64, 66, 72,74, 76, or 78.

Six of the candidate genes are members of a transcription factor genefamily based on the presence of a conserved domain (TCP domain). Thenucleotide and protein sequence alignments are shown in FIG. 3. Membersof this family are implicated in plant growth and developmentregulation. The conserved domain is thought to be responsible for DNAbinding to cis-elements in promoters in order to regulate downstreamgenes.

TABLE 5 Selected candidate putative gene sequences Contig Coding Nucleo-Protein SEQ ID NO Number Sequence tide (bp) (aa) (DNA/protein) C145337Predicted 867 288 39/40 C348 Predicted 2562 853 41/42 C131180 Predicted2790 929 43/44 C22266 Predicted 2478 825 45/46 C21860 Confirmed 1152 38347/48 C75660 Predicted 813 270 49/50 C11320 Predicted 762 253 51/52C1838 Predicted 753 250 53/54 G120126 Predicted 960 320 61/62 G151887Predicted 930 310 63/64 G135280 Predicted 822 274 65/66 G56830 Predicted1158 386 71/72 S4261 Predicted 1224 408 73/74 S950 Predicted 1014 33875/76 S1904 Predicted 1011 337 77/78

Example 5—Development of Transgenic Plants Containing RNAi orOver-Expression Constructs and Efficacy Testing

To investigate the function of the candidate genes, three sets oftransgenic plants were generated; a first using the full length codingsequence from tobacco (Table 4, SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,57, 59, or 69), a second using non-tobacco origin full length genes(Table 4, SEQ ID NOs: 55, 67, 79, or 81); and a third using a RNAisequence (SEQ ID NO: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, or 101). An expression vector, p45-2-7 (SEQ IDNO:112; FIG. 2), was used, which has a CsVMV promoter and a NOSterminator, as well as a cassette having a kanamycin selection marker(NPT II) under direction of the actin2 promoter and a NOS terminator.The nucleic acid constructs carrying the transgenes of interest wereintroduced into tobacco leaf discs using an Agrobacterium transformationapproach. See, for example, Mayo et al., 2006, Nat Protoc., 1(3):1105-11and Horsch et al., 1985, Science 227:1229-1231.

Briefly, tobacco plants (Narrow Leaf Madole (NLM)) were grown frommagenta boxes, and leaf disks were cut into 15×150 mm plates.Agrobacterium tumefaciens containing the target plasmid were collectedby centrifugation of 20 ml cell suspension in 50 ml centrifuge tube at3500 rpm for 10 minutes. Supernatant was removed and Agrobacterium cellpellet was resuspended in 40 ml liquid resuspension medium. About 25 mlof the solution was transferred to each 15×100 mm Petri plates. In those15×150 mm plates, tobacco leaves, avoiding the midrib, were cut into 0.6cm disk. Leaf disks were placed upside down, a thin layer of MS/B5liquid resuspension medium was added, and slices were made with a #15razor blade. The leaf discs were poked uniformly with a fine pointneedle. Eight disks were placed, upside down, in each regeneration plate(15×100 mm). Agrobacterium tumefaciens suspension was added and the leafdiscs were incubated for 10 minutes.

Leaf disks were transferred to co-cultivation plates (½ MS medium) anddisks were placed upside down in contact with filter paper overlaid onthe co-cultivation TOM medium (MS medium with 20 g sucrose/L; 1 mg/L IAAand 2.5 mg/L BAP). The plate was sealed with parafilm and labeledappropriately. Plates were incubated in dim light (60-80 mE/ms) and 18/6photoperiods at 24° C. for three days. Leaf disks were transferred toregeneration/selection TOM K medium plates (TOM medium with 300 mg/lKanamycin) and subculture bi-weekly to the same fresh medium in dimlight at 24° C. until shoots become excisable. Shoots from leaves wereremoved with forceps and inserted in MS basal medium with 100 mg/Lkanamycin at 24° C. and 18/6 photoperiods with light intensity of 6080mE/ms for rooting.

When plantlets with both shoots and roots grew large enough (e.g., reachover half of a GA7 box), they were transferred to soil foracclimatization. During the transfer, the gel was washed from the roottissue with tap water. Established seedlings were transferred to thegreenhouse for further analysis and to set seed.

Efficacy testing for sucker growth phenotypes were conducted by growingplants to laybe stage. These plants were topped and axillary bud growthwas observed at specific time points after topping.

FIG. 4A show a wild type plant (left) and a plant transformed with RNAconstruct #1 (SEQ ID NO:55; right) before topping, and FIG. 4B show thewild type plant (top) and the plant transformed with RNA construct #1(bottom) at the indicated times after topping. FIG. 4C shows the T1generation of wild type plants (left) and plants transformed with RNAconstruct #1 (right). The growth of the axillary buds after topping wasincreased substantially in the transgenic plants relative to the wildtype plants. Initiation of axillary bud growth in the transgenic plantswas already beginning even before the plant was topped, and the rate ofgrowth was increased for up to 1 week after topping. These resultsdemonstrated that the expression of RNA construct #1 is likelyresponsible for bud dormancy, and down-regulation of the gene is afactor in sucker initiation and growth.

The sequence of the expression cassette is shown in SEQ ID NO:111, withthe relevant portions indicated to the left.

Example 6—Promoter Cloning, Transformation and Analysis

The expression pattern of the 41 candidate genes were analyzed,promoters of the genes with high level expression in axillary bud, butlow expression levels in other tissues, were selected (Table 6).Expression pattern of these clones were confirmed by real-time PCRanalysis (FIG. 1). Six axillary meristem-specific promoters were clonedby PCR methods from TN90 genomic DNA using gene-specific primers. Thesequences of the promoters are shown in SEQ ID NO:113-118.

Expressions of candidate promoters were analyzed by transformation oftobacco with a chimeric candidate promoter::beta-glucuronidase (GUS)reporter gene with the same cassette described in Example 5. Thechimeric gene was introduced via Agrobacterium-mediated transformationinto a NLM line. Gus staining was used to locate promoter expressionfollowing the method of Crone et al., 2001, Plant Cell Environ.,24:869-874. Transgenic tobacco tissue was placed in cold 90% Acetone onice. When all samples were harvested, samples were placed at roomtemperature for 20 minutes. Acetone was removed from the samples, andstaining buffer (0.2% Triton X-100; 50 mM NaHPO4 Buffer, pH7.2; 2 mMPotassium Ferrocyanide) was added to samples, all the while keeping thesamples on ice. X-Gluc was added to the staining buffer to a finalconcentration of 2 mM—from a 100 mM stock solution of X-Gluc in DMF,which must be kept in the dark at −20° C. Staining buffer was removedfrom samples and fresh staining buffer with X-Gluc was added. Thesamples were infiltrated under vacuum, on ice, for 15 to 20 minutes. Thesamples were incubated at 37° C. from 2 hours to overnight. The sampleswere removed from the incubator and the staining buffer was removed.Samples were washed through an ethanol series (i.e., from 10%, 30%, 50%,70%; the sample can be heated to 60° C. to get rid of chloroplasts, ifdesired), to 95%, avoiding light, for 30 min each step. Finally, sampleswere kept in 100% ethanol.

The GUS-positive plant tissues were examined with a bright fieldmicroscope (Leica Q500MC, Cambridge, England) at a low magnification andphotographed with a digital camera. See FIGS. 5A and 5B. Results ofexperiments using two different promoters described herein (SEQ IDNOs:113 and 115) are shown in FIG. 5. Young seedlings were stained. TheGUS expression, indicated by the blue staining, is concentrated in theaxillary bud, indicating that these two promoters are active in theaxillary bud, but not in the stem or leaves (FIG. 5A). The expression ofGUS under the direction of the SEQ ID NO:113 promoter also decreasedafter topping, which coincides with the gene expression pattern that wasobserved for the endogenous gene that is normally regulated by thispromoter (FIG. 5B). These promoter sequences can be used to expressgenes only or predominantly in the axillary bud while limitingexpression in the rest of the plant.

TABLE 6 Selected clones for the promoter analysis Contig Length of SEQNumber Promoter ID NO C5787 2248 113 C7651 2800 114 C26207 3356 115C12866 3150 116 C41568 2964 117 C16249 941 118

Example 7—Efficacy Test of Promoter and Gene Combinations

After testing of the tissue-specific expression patterns of candidatepromoters using promoter::GUS fusion analysis in transgenic plants, weconstructed serial vectors to express the candidate genes only in theaxillary bud. Using Agrobacterium-mediated transformation, transgenicplants containing these constructs are generated. The expression of thecandidate gene(s) in the transgenic plants can result in the plantssuppressing axillary bud growth, resulting from either suppression orover-expression of candidate gene(s).

Some examples are shown as bellow:

Construct 1: Promoter (SEQ ID NO:113) and gene (SEQ ID NO: 17)

Construct 2: Promoter (SEQ ID NO:113) and gene (SEQ ID NO:104)

Construct 3: Promoter (SEQ ID NO:113) and gene (SEQ ID NO: 7)

Construct 4: Promoter (SEQ ID NO:113) and gene (SEQ ID NO: 41)

Construct 5: Promoter (SEQ ID NO:113) and gene (SEQ ID NO: 5)

Construct 6: Promoter (SEQ ID NO:118) and gene (SEQ ID NO: 17)

Construct 7: Promoter (SEQ ID NO:118) and gene (SEQ ID NO:104)

Construct 8: Promoter (SEQ ID NO:118) and gene (SEQ ID NO: 7)

Construct 9: Promoter (SEQ ID NO:118) and gene (SEQ ID NO: 41)

Construct 10: Promoter (SEQ ID NO:118) and gene (SEQ ID NO: 5)

Construct 11: Promoter (SEQ ID NO:115) and gene (SEQ ID NO: 17)

Construct 12: Promoter (SEQ ID NO:115) and gene (SEQ ID NO:104)

Construct 13: Promoter (SEQ ID NO:115) and gene (SEQ ID NO: 7)

Construct 14: Promoter (SEQ ID NO:115) and gene (SEQ ID NO: 41)

Construct 15: Promoter (SEQ ID NO:115) and gene (SEQ ID NO: 5)

Construct 16: Promoter (SEQ ID NO:117) and gene (SEQ ID NO: 17)

Construct 17: Promoter (SEQ ID NO:117) and gene (SEQ ID NO:104)

Construct 18: Promoter (SEQ ID NO:117) and gene (SEQ ID NO: 7)

Construct 19: Promoter (SEQ ID NO:117) and gene (SEQ ID NO: 41)

Construct 20: Promoter (SEQ ID NO:117) and gene (SEQ ID NO: 5)

Efficacy testing for the impact of constructs 1-20 will be carried outunder greenhouse and field conditions. Transgenic plants and matchedwild type controls will be grown to layby stage and topped. Suckergrowth will be quantified with a metric including the total number ofsuckers, the rate of sucker growth, and the emergence of new suckersafter sucker removal. These measurements will be conducted by hand or bydigital imaging technology. Field efficacy testing will also determinethe type and extent of sucker control chemical application needed undernormal agronomical practices. With this metric the effect of geneexpression constructs on axillary bud initiation and growth will becompared with wild type plants of the same variety. At the same time,the impact of this technology on costs related to sucker control and anychanges in chemical residues found in the final cured leaf will bequantified.

Example 8—TALEN-Mediated Mutagenesis

Transcription activator-like effector nucleases (TALENs) technology wasused to carry out genome modification in commercial tobacco varietiessuch as TN90, K326 and Narrow Leaf Madole. TALENs enable geneticmodification through induction of a double strand break (DSB) in a DNAtarget sequence. The ensuing DNA break repair by either non-homologousend joining (NHEJ) or homology-directed repair (HDR)-mediated pathwaycan be exploited to introduce the desired modification (e.g. genedisruption, gene correction or gene insertion).

To introduce TALENs and a donor DNA into a plant cell, PEG-mediatedprotoplast transformation was used. Tobacco leaves of 4-8 weeks oldtobacco plants from sterile culture were cut into small pieces andtransferred in a petri dish containing filter-sterilized enzyme solutionwith 1.0% Cellulase onuzuka R10 and 0.5% Macerozym. The leaf strips inthe petri dish were vacuum infiltrated for 30 min in the dark using adesiccator. After incubation, the digested leaves were resuspended byshaking at 45 rpm for 230 minutes and then filtered through a sterilizednylon filter (100 μm pore size) by collecting in a 50 ml centrifugetube. The solution laid on Lymphoprep was separated with thecentrifugation at 100 g for 10 min. The protoplast bands were collectedusing a Pasteur pipette, and purified protoplasts were washed with anequal volume of W5n solution containing with NaCl, CaCl₂), KCl, MES andGlucose, and centrifuged for 5 min at 2000 rpm. The protoplast pelletswere resuspended at 2×10⁵/ml in W5n solution, and left on ice for 30mins. Afterwards, the supernatant was removed and the protoplast pelletwas resuspended in filter-sterilized MMM solution containing mannitol,MgCl₂ and MES.

The PEG transfection of tobacco protoplasts was performed according tothe method described by Zhang et al. (2012) with some modifications. A500 μl aliquot of the protoplast suspension was transferred into 10 mlculture tube and 25 μl (10 μg) of plasmid DNA was added slowly to theprotoplasts suspension. In the protoplast-DNA solution, 525 μl PEGsolution was added, and mixed carefully by tapping the tube. The tubewas incubated for 20 minutes, then 2.5 ml W5n solution was added to stopthe reaction. The solution was centrifuged at 100 g for 5 min, andwashed with protoplast culture media. The PEG-treated protoplasts wereresuspended in 1 ml culture media containing with 0.1 mg/l NAA and 0.5mg/l BAP, and mixed with 1 ml low-melting agar to make protoplast beads.The protoplast beads were cultured in liquid media, and calli growingfrom the protoplast beads were transferred onto solid shooting media.When shoots were well developed, the shoots were transferred in amagenta box for root formation.

When root systems were fully developed and shoot growth had resumed,plants were transplanted into soil.

TALEN approaches that can be used to prevent or reduce sucker growthinclude: (1) for targeted genomic integration in tobacco varieties,gene-specific TALENs and a donor DNA with homology-derived recombination(HDR) are designed; (2) for sucker-specific promoter and target geneinsertion in the tobacco genome; and (3) for target gene disruption,gene-specific TALENs with, e.g., non-homologous end joining (NHEJ) areused to direct the TALENs to the target gene disruption.

(1) Targeted Genomic Integration:

(A) Targeted Genomic Integration of a Coding Sequence into the PromoterRegion of a Gene with Highly Specific Expression in Axillary Bud:

Instead of random gene insertion using conventional transformationmethods, the targeted genomic integration of a coding sequence into thepromoter region of a gene with highly specific expression in axillarybud can be used to control the expression of the coding sequence by theendogenous promoter activity. One example of the targeted genomicintegration approach is the combination of a promoter (SEQ ID NO:118)and a coding sequence (SEQ ID NO:1). Using such a construct, a codingsequence (or more than one coding sequence) is homologously recombinedinto the genomic region of the promoter sequence and controlled by thepromoter.

A TALEN donor sequence is shown in SEQ ID NO:119 (the promoter andtarget sequences are underlined, and the target gene sequence is inbold), and a TALEN target sequence is shown in SEQ ID NO:120 (the targetsequences are underlined).

(B) Targeted Genomic Integration of a Promoter and a Coding Sequenceinto the Promoter Region of a Gene with Highly Specific Expression inAxillary Bud:

To effectively provide a double dose of promoter control, asucker-specific promoter and a coding sequence can be inserted into thepromoter region of a gene highly expressed in axillary bud. In thisapproach, two promoters work together to control the coding sequence (orcoding sequences). For example, in one end of a promoter (SEQ IDNO:118), a construct including a promoter (SEQ ID NO:113) and a codingsequence (SEQ ID NO:13) is inserted using TALEN technology, therebydirecting expression of the coding sequence by both promoters (SEQ IDNO:118 and 113).

A TALEN donor sequence is shown in SEQ ID NO:121 (the endogenouspromoter is underlined, the exogenous promoter is italicized, and thetarget gene is in bold).

(C) Sucker-Specific Promoter and Coding Sequence Insertion

Another option of targeted gene integration is to insert a selectedtobacco promoter and coding sequence into an effective location of thetobacco genome by TALEN.

(2) Target Gene Disruption

To disrupt the function of candidate genes without using RNAiconstructs, gene-specific TALENs were designed and introduced intotobacco cells, resulting in deletions or insertions to knockout theendogenous gene (or genes). For example, potential TALEN target sites ina coding sequence (SEQ ID NO:104) were identified, and homologousrecombination sites within the coding sequence of the gene wereselected.

A TALEN target sequence is shown in SEQ ID NO:122 (the target sequencesare underlined).

Example 9—Additional Transgenic Strategies

The following strategies to regulate sucker outgrowth are describedherein.

The first strategy applied was to regulate axillary bud outgrowth geneexpression. Mutant studies in Arabidopsis, rice, and barley suggest thatthe genetic pathways that regulate branching are complex. There are twogeneral classes of genes that regulate branching. One class of genesrestricts the out-growth of buds and is defined by mutants withincreased branching. See, for example, the Arabidopsis BRANCHED1 gene(e.g., SEQ ID NO:81 and the possible tobacco homologs shown in SEQ IDNOs: 1, 13, 35, 37, 39) and the Arabidopsis More Axillary Branching(MAX) gene. The other class of genes promotes axillary meristemdevelopment and is defined by mutants with decreased branching. See, forexample, the Arabidopsis Lateral Suppressor (LAS) gene and the possiblehomologues in tobacco (e.g., SEQ ID NOs: 71 or 73) as well as theArabidopsis Regulator of Axillary Meristems (RAX) and the possibletobacco homologous (e.g., SEQ ID NOs: 75 and 77).

The second strategy applied was to regulate tobacco cytokinin synthesisand distribution. As a plant hormone, cytokinin plays many regulatoryroles in shoot growth, retardation of leaf senescence, inhibition ofroot growth, seed germination, and stress responses. It is well-knownthat cytokinin promotes axillary bud outgrowth. When cytokinin isapplied directly to axillary buds or supplied via the xylem stream, sidebranches are increased. Cytokinin oxidase/dehydrogenase (CKX) is anenzyme that degrades cytokinin. Overexpression of individual CKX genesestablished cytokinin deficient plants and revealed that cytokinin is apositive regulator of the shoot meristem activity. On the other hand,reduced expression of CKX in rice causes cytokinin accumulation in shootmeristems, which increases the number of buds such as floral buds,ultimately resulting in enhanced grain yield. Based on these results,CKX expression in axillary buds can inhibit or delay axillary budoutgrowth in tobacco after the shoot apical meristem has been topped.

Decapitation of the shoot apex releases axillary buds from theirdormancy and they begin to grow out. Auxin derived from an intact shootapex suppresses axillary bud outgrowth, whereas cytokinin induced bydecapitation of the shoot apex stimulates axillary bud outgrowth.Depletion of cytokinin in the axillary bud region by overexpression ofthe relevant enzymes under control of an axillary bud specific promotercan be used to inhibit axillary meristem outgrowth. The candidate genesinvolved in this strategy are Arabidopsis cytokinin oxidase (CKX; SEQ IDNO:55 encoding SEQ ID NO:56); tobacco CKXs (SEQ ID NOs:57, 59, or 61);and tobacco adenosine phosphate-isopentenyltransferase (IPT) (SEQ ID NO:61).

The third strategy applied was to control axillary bud outgrowth bydestroying axillary apical meristem development. There are two types ofthe expression of transgenes in transgenic plants: constitutiveexpression and tissue specific expression. The constitutive geneexpression can result in unexpected problems if a gene of criticalimportance in a certain tissue is miss-expressed in other tissues.Unlike constitutive expression of genes, tissue-specific expression isthe result of gene regulation in a target tissue. For tissue-specificexpression, promoters can control the expression of given genes in atissue-dependent manner and according to the developmental stage of theplant. In our case, the promoters were obtained from genes specificallyexpressed in axillary buds, and the promoters were defined to regulategene expression in buds. To control sucker growth after topping of ashoot apical meristem, the promoters (or modified promoters) can be usedto direct expression of a heterologous gene in tobacco plants. As aresult of axillary bud-specific expression, the heterologous gene (orthe transgene) operably linked to the promoter is expressed in axillarybuds where expression of the transgene is desired, leaving the rest ofthe plant unaffected by transgene expression.

Shoot meristems of plants are composed of stem cells that arecontinuously replenished through a classical feedback circuit involvingthe homeobox WUSCHEL (WUS) gene and the CLAVATA (CLV) gene signalingpathway. Targeting of the WUSHEL sequence or overexpression of theCLAVATA gene in axillary buds alters the pathway and causes a defect inshoot meristem development and inhibits shoot outgrowth. The candidategenes are WUS (SEQ ID NOs: 63 and 65) and CLV3 (SEQ ID NO: 67).

The CENTRORADIALIS (CEN) gene, which is required for indeterminategrowth of the shoot meristem in Antirrhinum, was cloned andcharacterized. When the gene is expressed in tobacco, the tobacco plantsshowed an extended vegetative phase, delaying the switch to flowering.In tobacco, the CET genes (from “CEN-like genes from tobacco”) are notexpressed in the main shoot meristem; their expression is restricted tovegetative axillary meristems. It is clear that CET genes play a role inthe development of vegetative axillary meristems to axillary bud growth,however, their actual function remains unknown. When their expression issilenced using an RNAi_CET construct, the transgenic plants show budgrowth retardation after topping.

Example 10—Experimental Data

Plant tissues were stained for GUS by immersion in a staining solution(50 mM sodium phosphate buffer, pH 7.0, 1 mM EDTA, 0.5 mg/mL5-bromo-4-chloro-3-indolyl-D GlcUA [X-Gluc; Biosynth AG], 0.4% TritonX-100, and 5 mM each of potassium ferri/ferrocyanide), and incubated at37° C. for 6-24 h.

The promoter shown in SEQ ID NO:117 has been shown to be a goodcandidate for specific expression in the axillary bud before topping andfor 15 days after topping. There was no expression in the shoot apicalmeristem region before topping. The promoter shown in SEQ ID NO:117(about 2.5 kb) is the 5′ end upstream of sequence of SEQ ID NO: 27,which encodes eukaryotic translation initiation factor 3, subunit A(eIF-3A), a component of the eukaryotic translation initiation factor 3(eIF-3) complex, which is required for several steps in the initiationof protein synthesis.

Several genes were stacked by co-transformation to overexpress and/orknock down using, for example, RNAi, under the control of the promotershown in SEQ ID NO:117. The following are the constructs and genes thatwere stacked together by co-transformation.

-   -   a) Promoter SEQ ID NO:117-RNAi_CET2-26-6, which targets CET2,        SEQ ID NO: 11 and SEQ ID NO:49;    -   b) Promoter SEQ ID NO:117-RNAi_CET2-26-6, co-transformed with        Promoter SEQ ID NO:117-AtBRC1 (SEQ ID NO:81);    -   c) Promoter SEQ ID NO:117-RNAi_CET2-26-6, co-transformed with        Promoter SEQ ID NO:117-SEQ ID NO: 1; and    -   d) Promoter SEQ ID NO:117-SEQ ID NO: 1, co-transformed with        Promoter SEQ ID NO:117-AtBRC1 (SEQ ID NO:81).

It is to be understood that, while the methods and compositions ofmatter have been described herein in conjunction with a number ofdifferent aspects, the foregoing description of the various aspects isintended to illustrate and not limit the scope of the methods andcompositions of matter. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1.-39. (canceled)
 40. A method of producing a tobacco product, themethod comprising: (a) providing a leaf from a tobacco plant comprisinga heterologous nucleic acid molecule operably linked to a promotercomprising a sequence selected from the group consisting of SEQ ID NOs:113-118, wherein the cured leaf comprises the heterologous nucleic acidmolecule; and (b) manufacturing the tobacco product using the leaf. 41.The method of claim 40, wherein the leaf is a cured leaf.
 42. The methodof claim 41, wherein the cured leaf is selected from the groupconsisting of an air cured leaf, a fire cured leaf, a flue cured leaf,and a sun cured leaf.
 43. The method of claim 40, wherein the leaf isselected from the group consisting of an aged leaf, a conditioned leaf,and a fermented leaf.
 44. The method of claim 43, wherein the aged leafis aged for 2 years to 5 years.
 45. The method of claim 41, wherein thecured leaf is further subjected to a process selected from the groupconsisting of aging, conditioning, and fermenting.
 46. The method ofclaim 40, wherein the heterologous nucleic acid comprises a nucleic acidsequence encoding a polypeptide having at least 90% sequence identity toan amino acid sequence encoded by a polynucleotide sequence of SEQ IDNO:
 79. 47. The method of claim 40, wherein the heterologous nucleicacid comprises a nucleic acid sequence encoding a polypeptide having atleast 95% sequence identity to an amino acid sequence encoded by apolynucleotide sequence of SEQ ID NO:
 79. 48. The method of claim 40,wherein the heterologous nucleic acid comprises a nucleic acid sequenceencoding a polypeptide having 100% sequence identity to an amino acidsequence encoded by a polynucleotide sequence of SEQ ID NO:
 79. 49. Themethod of claim 40, wherein the heterologous nucleic acid moleculeencodes a polypeptide having at least 98% identity to a polypeptidesequence of SEQ ID NO:
 80. 50. The method of claim 40, wherein theheterologous nucleic acid molecule encodes a polypeptide having 100%identity to a polypeptide sequence of SEQ ID NO:
 80. 51. The method ofclaim 40, wherein the tobacco plant is selected from the groupconsisting of a Burley type, a dark type, a flue-cured type, a Marylandtype, and an Oriental type.
 52. The method of claim 40, wherein thetobacco plant is a variety selected from the group consisting of BU 64,CC 101, CC 200, CC 13, CC 27, CC 33, CC 35, CC 37, CC 65, CC 67, CC 301,CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, CC 1063, Coker 176,Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, GL 26H, GL 338, GL350, GL 395, GL 600, GL 737, GL 939, GL 973, GF 157, GF 318, RJR 901, HB04P, K 149, K 326, K 346, K 358, K394, K 399, K 730, NC 196, NC 37NF, NC471, NC 55, NC 92, NC2326, NC 95, NC 925, PVH 1118, PVH 1452, PVH 2110,PVH 2254, PVH 2275, VA 116, VA 119, KDH 959, KT 200, KT204LC, KY 10, KY14, KY 160, KY 17, KY 171, KY 907, KY907LC, KTY14×L8 LC, LittleCrittenden, McNair 373, McNair 944, msKY 14×L8, Narrow Leaf Madole, NC100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6,NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal SmithMadole, OXFORD 207, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R7-11, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168,Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20,Speight NF3, TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN90LC, TN 97,TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, or VA359. 53.The method of claim 40, wherein the tobacco product is selected from thegroup consisting of cigarillos, non-ventilated recess filter cigarettes,vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigartobacco, cigarette tobacco, chewing tobacco, leaf tobacco shreddedtobacco, cut tobacco, snuff, long-cut moist smokeless tobacco, and snus.54. The method of claim 40, wherein the tobacco product is a smokelesstobacco product.
 55. The method of claim 40, wherein the leaf isprocessed in a manner selected from the group consisting of being cut,expanded, blended, milled, or comminuted.
 56. The method of claim 40,wherein the tobacco product further comprises an ingredient selectedfrom the group consisting of a binder, a plasticizer, a stabilizer, anda flavoring.
 57. The method of claim 40, wherein the tobacco plantexhibits reduced axillary bud growth after topping as compared to acontrol tobacco plant not expressing the heterologous nucleic acidmolecule.